Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus.
Stem cells are divided into embryonic stem cells(ES cells) and tissue-specific stem cells depending on their differentiation potencies. ES cells are isolated from the inner cell mass(ICM) of embryos at the blastocyst stage and are pluripotent, i.e., they are capable of differentiating into virtually every type of cells found in an organism.
In contrast, tissue-specific stem cells appear at a stage of organ formation during the embryonic development and they are organ-specific and multipotent, i.e., they are generally committed to give rise to cells constituting a specific organ. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes; and mesenchymal stem cells, to the cells of connective tissues, e.g., osteoblasts, chondroblasts, adipocytes and myoblasts.
Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of human embryonic stem cell. The most noticeable potential application of the stem cells is their use as a perfect source of cell supply for a cell replacement therapy. Hardly curable diseases, e.g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ, and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy.
However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method, wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem that it is difficult to obtain a sufficient amount of cells to be supplied to the patient. In order to solve this problem, cells of a specific tissue differentiated from an isolated embryonic stem cell or differentiated cells from isolated and proliferated tissue-specific stem cells can be employed in a cell replacement therapy.
Hitherto, it has been proven that mouse embryonic stem cells can be differentiated on a culture dish into various cells such as hematopoietic cells, myocardial cells, insulin-secreting pancreatic cells and neural cells. Further, several reports have demonstrated that transplantation of the cells differentiated from stem cells is effective in the treatment of a disease caused by the loss of cells. For instance, synthesis of myelin in a mouse increased when myelin-synthesizing oligodendrocytes differentiated from an embryonic stem cell were transplanted into the mouse(Brustle et al., Science, 285: 754–756, 1999). Blood sugar level was regulated by transplanting insulin-secreting cells differentiated from an embryonic stem cell into a diabetes mouse model (Soria et al., Diabetes, 49: 157–162, 2000). Further, dyscinesia caused by spinal cord injury was remedied significantly by transplanting neural cells differentiated from an embryonic stem cell into a mouse having spinal cord injury(McDonald et al., Nat. Med., 5(12): 1410–1412, 1999).
However, since the human embryonic stem cell has been successfully isolated only recently and there is no report on the differentiation of the embryonic stem cell on a culture dish into other specific cells than neural cells, clinical use of specific tissue cells differentiated from an embryonic stem cell in a cell replacement therapy still remains on a level of possibility.
Further, since the efficiency of differentiation from an embryonic stem cell into target cells is low, there is a risk of an adverse side effect caused by other cells mixed with the target cells during transplantation. Accordingly, there exists a need for the development of a precise differentiation method for safer clinical application of the cells differentiated from embryonic stem cells.
On the other hand, in case when tissue-specific stem cells are employed in a cell replacement therapy, there is the problem that lowering of the proliferating ability of the cells or differentiation into unfavorable cells may occur during a long-term culture. Further, transplantation of neural cells is required for the treatment of a neurodegenerative disease such as Parkinson's disease. Since it is difficult to obtain neural stem cells directly from the patient, they are generally obtained by culturing neural stem cells isolated from the brain tissue of dead fetus and differentiating them into neural cells. However, the use of fetal brain invites the ethical problem as well as is limited by insufficient supply, and may cause an immunological rejection. Further, most of neural stem cells are liable to differentiate into astrocytes rather than neurons.
Accordingly, if it is possible to differentiate mesenchymal stem cells in patient's own bone marrow into neural cells to be used in a cell replacement therapy, neural cells can be readily supplied and such problems as immunological rejection would not occur during treatment.
Hitherto, it has been considered that one kind of stem cells differentiate only into the cells of a tissue belonging to a specific system. It was reported that mesenchymal stem cells formed in vitro colonies in the presence of various growth factors such as platelet-derived growth factor, basic fibroblast growth factor(bFGF), transforming growth factor-β (TGF-β) and epidermal growth factor(EGF)(Kuznetsov et al., Br. J. Haematol., 97: 561, 1997; and van den Bos C. et al., Human Cell, 10:45, 1997), and about one-third of initially attached cells had a multipotency, thereby differentiating into connective tissue cells such as osteoblasts, chondroblasts and adipocytes(Pittenger M F et al., Science, 284: 143, 1999). Further, Ferrari G. et al. reported that bone marrow is a source of myogenic precursor cells that form new muscles(Science, 279: 1528, 1998).
Recent studies reported that mesenchymal stem cells can also differentiate into the cells of the neural system. For instance, Sanchez-Ramos et al. reported that mesenchymal stem cells differentiated into neurons and astrocytes upon culture in the presence of retinoic acid and brain-derived neurotrophic factor(BDNF)(Exp. Neurology, 164: 247–256, 2000). Dale Woodbury et al. reported that mesenchymal stem cells in bone marrow differentiated into neural cells in the presence of antioxidants such as β-mercaptoethanol and dimethyl sulfoxide(DMSO)(J. Neuro. Res., 61: 364–370, 2000). However, the use of strong differentiation-inducing agents such as DMSO may cause a problem in a clinical application.
The present inventors have endeavored to discover materials that are highly safe and capable of differentiating stem cells in bone marrow into neural cells, and have discovered that HGF promotes the differentiation of mesenchymal stem cells of bone marrow into neural cells, and also that the addition of EGF and bFGF to a culture medium, together with HGF, significantly enhances both the differentiation of stem cells into neural cells and the amplification of the resulting neural cells.
It has been reported that EGF and bFGF stimulate the differentiation of neural stem cells into neurons or astrocytes when they are added to a serum-free medium for culturing neural stem cells separated from the brain tissue (Melissa et al., Exp. Neurology, 158: 265–278, 1999).
HGF has been reported to enhance the viability of neurons in hippocampus and mesencephalon, and induce the growth of neurite in neocortical explant(Hamanoue M et al., J. Neurosci. Res., 43: 554–564, 1996). Further, in the peripheral nervous system, it functions as an existence factor for motoneurons(Ebens A et al., Neuron, 17: 1157–1172, 1996), and involves in the growth and existence of sensory neurons and parasympathetic neurons (Fleur Davey et al., Mol. Cell Neurosci., 15: 79–87, 2000).
However, it has never been reported that mesenchymal stem cells can be differentiated into neural cells by culturing them in a medium containing EGF, bFGF and HGF.